Closure of the Venus flytrap module of mGlu8 receptor and the activation process: Insights from mutations converting antagonists into agonists

doi:10.1073/pnas.162138699

Closure of the Venus flytrap module of mGlu8 receptor and the activation process: Insights from mutations converting antagonists into agonists

^*Départment de Chimie et Biochimie Pharmacologiques et Toxicologiques, Unité Mixte de Recherche 8601–Centre National de la Recherche Scientifique, Université René Descartes-Paris V, 45 Rue des Saints-Pères, 75270 Paris Cedex 06, France; and^† Départment de Mécanismes Moléculaires des Communications Cellulaires, Unité Propre de Recherche 9023–Centre National de la Recherche Scientifique, 141 Rue de la Cardonille, 34094 Montpellier Cedex 5, France

Edited by Henry R. Bourne, University of California, San Francisco, CA, and approved June 7, 2002 (received for review March 11, 2002)

Abstract

Ca²⁺, pheromones, sweet taste compounds, and the main neurotransmitters glutamate and γ-aminobutyric acid activate G protein-coupled receptors (GPCRs) that constitute the GPCR family 3. These receptors are dimers, and each subunit has a large extracellular domain called a Venus flytrap module (VFTM), where agonists bind. This module is connected to a heptahelical domain that activates G proteins. Recently, the structure of the dimer of mGlu1 VFTMs revealed two important conformational changes resulting from glutamate binding. First, agonists can stabilize a closed state of at least one VFTM in the dimer. Second, the relative orientation of the two VFTMs in the dimer is different in the presence of glutamate, such that their C-terminal ends (which are connected to the G protein-activating heptahelical domain) become closer by more than 20 Å. This latter change in orientation has been proposed to play a key role in receptor activation. To elucidate the respective role of VFTM closure and the change in orientation of the VFTMs in family 3 GPCR activation, we analyzed the mechanism of action of the mGlu8 receptor antagonists ACPT-II and MAP4. Molecular modeling studies suggest that these two compounds prevent the closure of the mGlu8 VFTM because of ionic and steric hindrance, respectively. We show here that the replacement of the residues responsible for these hindrances (Asp-309 and Tyr-227, respectively) by Ala allows ACPT-II or MAP4 to fully activate the receptors. These data are consistent with the requirement of the VFTM closure for family 3 GPCR activation.

Footnotes

↵ ‡ To whom reprint requests should be addressed. E-mail: pin{at}montp.inserm.fr.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:
1. ACPT-I, (1R,3S,4R)-1-aminocyclopentane-1,3,4-tricarboxylic acid
2. ACPT-II, (1R,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid
3. DCG-IV, (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine
4. GPCR, G protein-coupled receptor
5. HD, heptahelical domain
6. L-AP4, (S)-2-amino-4-phosphonobutanoic acid
7. LY341495, (αS)-α-amino-α-[(1S,2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid
8. MAP4, (S)-2-amino-2-methyl-4-phosphonobutanoic acid
9. MCPG, α-methyl-4-carboxyphenylglycine
10. mGlu, metabotropic glutamate
11. VFTM, Venus flytrap module
12. 3D, three-dimensional
13. wt, wild type
14. HA, hemagglutinin
15. IP, inositol phosphate